Crystalline form of 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride

ABSTRACT

The present invention is directed to a novel crystalline hydrate of 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]-phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride and uses for same, including inhibition of disease states associated with estrogen deprivation including cardiovascular disease, hyperlipidemia, and osteoporosis; and inhibition of other pathological conditions such as endometriosis, uterine fibrosis, estrogen-dependent cancer (including breast and uterine cancer), prostate cancer, benign prostatic hyperplasia, CNS disorders including Alzheimer&#39;s disease, prevention of breast cancer, and up-regulating ChAT.

This application claims the benefit under Title 35, U.S.C. §120 of PCTInternational Patent Application No. PCT/US00/16332, filed Jul. 17,2000, which claims priority under Title 35 U.S.C. §119(e) of ApplicationNos. 60/146,184, filed Jul. 29, 1999; 60/147,642, filed Aug. 6, 1999;and 60/149,820 filed Aug. 19, 1999.

BACKGROUND OF THE INVENTION

6-Hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophenehydrochloride (arzoxifene) was first described generically in U.S. Pat.No. 5,510,357 and was specifically disclosed in U.S. Pat. No. 5,723,474('474) and European Patent Application 0729956. Arzoxifene is anonsteroidal mixed estrogen antagonist/agonist, useful for, inter alia,lowering serum cholesterol and for inhibiting hyperlipidemia,osteoporosis, estrogen dependent cancers including breast and uterinecancer, endometriosis, CNS disorders including Alzheimer's disease,aortal smooth muscle cell proliferation, and restenosis.

Specifically, arzoxifene is useful for, and is being clinicallyevaluated for the treatment of receptor positive metastatic breastcancer; the adjuvent treatment of receptor positive patients followingappropriate systemic or local therapy; the reduction of recurrence ofinvasive and noninvasive breast cancer; and the reduction of theincidence of invasive breast cancer and ductal carcinoma in situ (DCIS).Arzoxifene is also useful in combination with radiotherapy, aromataseinhibitors, LHRH analogues, and acetyl choline esterase (AChE)inhibitors.

X-ray powder diffraction (XRD), thermogravimetric (TGA), proton nuclearmagnetic resonance (¹H NMR) and Karl Fischer (KF) analyses of bulkarzoxifene isolated by the procedures taught in '474 later indicatedthat said material was hydrated, poorly crystalline, and containedvariable amounts of an organic volatile (ethyl acetate) in its lattice.

Poorly crystalline materials are typically less desirable than highlycrystalline materials for formulation processing. In addition, it isgenerally not desirable to formulate pharmaceuticals containingsubstantial amounts of organic solvent (e.g., ethyl acetate) due topotential solvent toxicity to the recipient thereof and changes inpotency of the pharmaceutical as a function of the solvent.

Although the arzoxifene prepared by the procedures taught in '474 couldbe used as a pharmaceutical it would be highly desired and advantageousto find a more crystalline form of arzoxifene that did not contain anorganic solvent within its crystal lattice which could be reproduciblyand efficiently prepared on a commercial scale.

SUMMARY OF THE INVENTION

The present invention is related to a novel non-stoichiometric hydratedcrystalline form of6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophenehydrochloride (F-III) having an X-ray diffraction pattern whichcomprises the following peaks: 4.6±0.2, 7.8±0.2, 9.3±0.2, 14.0±0.2,17.6±0.2, 20.8±0.2, and 24.3±0.2° in 2θ; when obtained at 25±2° C. and35±10% relative humidity (RH) from a copper radiation source.

Moreover the present invention relates to a pharmaceutical formulationcomprising F-III; one or more pharmaceutical carriers, diluents, orexcipients; and optionally estrogen, optionally progestin, optionally anaromatase inhibitor, optionally an LHRH analogue and optionally anacetyl choline esterase (AChE) inhibitor.

In addition, the present invention is related to methods for using F-IIIto inhibit pathological conditions such as: uterine fibrosis,endometriosis, aortal smooth muscle cell proliferation, restenosis,breast cancer, uterine cancer, prostatic cancer, benign prostatichyperplasia, bone loss, osteoporosis, cardiovascular disease,hyperlipidemia, CNS disorders, and Alzheimer's disease and for usingF-III for the manufacture of a medicament for inhibiting same.

The present invention is further related to methods for using F-III toup-regulate choline acetyltransferase (ChAT) and for using F-III for themanufacture of a medicament for up-regulating same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representative differential scanning calorimetry (DSC)/TGAtrace of S-II.

FIG. 2 is a representative DSC/TGA trace of F-I.

FIG. 3 is a representative DSC/TGA trace of F-III.

FIG. 4 depicts moisture sorption isotherms for F-I and F-III.

FIG. 5 depicts desolvation of S-II as a function of drying time andtemperature.

DETAILED DESCRIPTION OF THE INVENTION

Bulk arzoxifene prepared by the procedure taught in '474 (Example 41,crystallization from a mixture of ethanol and ethyl acetate, filtrationand drying of the filter cake in vacuo to a constant weight at roomtemperature) was characterized by XRD and was found to be poorlycrystalline. ¹H NMR confirmed that the bulk material contained 6% ethylacetate.

The crystallization procedure taught in '474 was subsequently modifiedso that ethanol was added to a suspension of crude arzoxifene inrefluxing ethyl acetate. Upon cooling and vacuum filtration, the solidthat results from this modified procedure is a highly crystalline mixedethyl acetate/water solvate of arzoxifene (hereinafter referred to asS-II) which was later discovered to be a starting material for F-I(another non-stoichiometric hydrated crystalline form of arzoxifene).

F-I may be prepared by removing the ethyl acetate from S-II's crystallattice by vacuum drying/annealing S-II at elevated temperatures. Thetime and temperature required to anneal S-II in order to prepare F-Iwill vary from lot to lot but is typically on the order of 5 days ataround 100° C. High temperatures are needed to effect the conversion ofS-II to F-I via this procedure, since slurrying S-II in water at ambienttemperature or storing a sample at 98% RH for 3 weeks afforded noconversion to F-I. Furthermore, drying S-II in a convection oven at hightemperatures did not desolvate the material either, suggesting that avacuum is also required to pull the ethyl acetate from S-II's lattice.Preferably, F-I is readily prepared and isolated at ambient temperatureby crystallization of arzoxifene (or any polymorph/solvate thereof) fromtetrahydrofuran.

In accordance with the present invention, a particularly preferred formof arzoxifene is F-III. F-III is readily prepared and isolated atambient temperature. Only moderate drying conditions are required toremove low levels of residual crystallization solvent in the preparationof F-III. These moderate drying conditions consistently result in asolid of high purity (i.e., free of residual organic solvent) andcrystallinity and, thus, use of F-III eliminates toxicology issuesassociated with residual and crystal lattice organic solvent.Furthermore, preparation of F-III is simple and efficient, i.e., isamenable to bulk manufacture.

F-III is readily prepared and isolated at ambient temperature bycrystallization of arzoxifene (or any polymorph/solvate thereof) from amixture of isopropyl alcohol (IPA) and water. Typically, arzoxifene maybe suspended in a mixture of IPA and water and heated in order to effectdissolution of the arzoxifene starting material. Once dissolution isachieved, the solution is allowed to cool slowly to room temperature andthen further (with the aid of an ice bath or refrigeration) to between 0and 5° C. After a sufficient amount of time has elapsed forcrystallization to occur, the crystals may be collected by vacuumfiltration and dried to a constant weight in vacuo to obtain F-III inyields greater than 80%.

Suitable arzoxifene starting material for the above crystallizationincludes, but is not limited to, S-II, F-I, arzoxifene prepared by theprocedures taught in '474, or any mixture thereof. It is not importantwhich form of arzoxifene one starts with because crystallization fromIPA and water, according to the procedures described herein, results inF-III crystals. The ratio of water to IPA (v:v) is generally about 1:1to 9:1. More preferably, the ratio is between 2.5 and 5.6:1. Mostpreferably, the ratio is between 3 to 5.6:1. Upon collection of thecrystals by vacuum filtration, the F-III wet cake may be washed withcold deionized water before drying in vacuo. In addition, slightlyelevated drying temperatures (about 50° C. for 12 to 24 hours) arepreferred. For commercial scale synthesis of F-III, it may beadvantageous to seed the crystallization with F-III.

In a preferred process, F-III is prepared, isolated, and purifiedcontiguous with the chemical removal of the 6-isopropyl hydroxyprotecting group from6-isopropoxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophenehydrochloride (precursor A). The deprotection reaction is monitored forcomplete removal of the isopropyl protecting group and once it isdetermined that the removal is substantially complete, the work-up ofthe reaction will preferably include a crystallization under theconditions that provide F-III as discussed above and below. Methods forpreparing precursor A and for removing the isopropyl group may be foundin U.S. Pat. No. 5,723,474, the teachings of which are hereinincorporated by reference.

In another preferred process, F-III is prepared, isolated and purifiedcontiguous with the chemical reduction of the S-oxide and chemicalremoval of the benzyl protecting group from the 6-hydroxyl in6-benzyloxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene-(S-oxide)(precursor B). The reduction and deprotection reactions are monitoredfor complete reduction of the sulfoxide to the sulfide and completeremoval of the benzyl hydroxy protecting group. Once it is determinedthat the reduction/removal is substantially complete, the work-up of thereaction will preferably include a crystallization under the conditionsthat provide F-III as discussed herein. Methods for preparing precursorB, for removing the benzyl group, and for reducing the 1-sulfoxide tothe corresponding sulfide may also be found in the previouslyincorporated by reference U.S. Pat. No. 5,723,474.

Irrespective of the chemistry utilized in the deprotection and reductionsteps, crystallization of arzoxifene from the IPA/water solutionsdisclosed herein consistently produces F-III crystals in high purity.

Characterization and Differentiation of S-II, F-I and F-III

DSC/TGA and XRD methods were used to characterize S-II, F-I and F-III.TGA is often very useful for distinguishing between different solidforms of a material because the temperature(s) at which a physicalchange in a material occurs is usually characteristic of the polymorphor solvate. DSC is a technique that is often used to screen compoundsfor polymorphism and solvate formation. Lastly, XRD is a technique thatdetects long-range order in a crystalline material.

Arzoxifene prepared by the procedures taught in '474 gave XRD patternswith poor signal-to-noise ratios and a raised baseline, indicative ofpoorly crystalline material. Therefore, comparisons of F-I and F-III aremade to the material (S-II) produced by the modified arzoxifenecrystallization procedure discussed above (addition of ethanol to asuspension of arzoxifene in refluxing ethyl acetate).

Representative DSC/TGA traces of S-II, F-I and F-III are shown in FIGS.1, 2 and 3, respectively. The DSC trace for S-II shows a broad endothermbeginning at about 62° C., corresponding to the loss of ethyl acetateand water from the lattice. The endotherm beginning at about 152° C.represents a melt. The TGA weight loss of approximately 2.5% occurssimultaneous with the first transition, while the remaining 0.5% weightloss occurs up to the onset of melting, suggesting that some solventmolecules are more tightly held in the lattice.

The DSC trace of F-I shows a broad endotherm beginning at about 75° C.,followed by a second endotherm beginning at about 155° C. correspondingto a melt. The TGA trace of F-I shows a gradual weight loss of 0.3%followed by a sharp loss of 1.5%, which together represent dehydrationof the lattice. The onset of the first DSC transition and thecorresponding TGA weight loss are offset slightly due to the differencein heating rates. The initial weight loss represents weakly held watersof hydration while the second weight loss is consistent withapproximately 0.5 mole of water present in the lattice at very lowrelative humidities (below 5%—see moisture sorption data).

The DSC trace of F-III features a broad, low temperature endotherm atabout 30° C., followed by a second broad and relatively weak endothermbeginning at about 70° C., and a final transition beginning at about146° C. corresponding to a melt. The sharp 1.5% (˜0.5 mole) weight lossin the TGA coincident with the first endotherm corresponds to loss ofweakly held water molecules, while the additional ˜1.6% weight lossabove 60° C. represents loss of more tightly held water molecules, i.e.,those which are present at very low relative humidities. The weight lossobserved after 170° C. corresponds to decomposition of F-III.

The XRD patterns of F-I and F-III feature sharp peaks and a flatbaseline, indicative of highly crystalline materials. The angular peakpositions in 2θ and corresponding I/I_(o) data for representativesamples of F-I, F-III and S-II is tabulated in Table 1. Although many ofthe intense reflections are generally at similar diffraction angles,each of the forms gives a different powder pattern, allowing for a cleardistinction between S-II, F-I and F-III.

It is well known in the crystallography art that, for any givenpolymorph, the relative intensities of the diffraction peaks may varydue to preferred orientation resulting from factors such as crystalmorphology. Where the effects of preferred orientation are present, peakintensities are altered, but the characteristic peak positions of thepolymorph are unchanged. See, e.g., The United States Pharmacopeia #23,National Formulary #18, pages 1843-1844, 1995. Thus, based on peakintensities as well as peak position, F-III may be identified by thepresence of peaks at 4.6±0.2, 7.8±0.2, 9.3±0.2, 14.0±0.2, 17.6±0.2,20.8±0.2, and 24.3±0.2° in 2θ; when the pattern is obtained at 25±2° C.and 35±10% relative humidity from a copper radiation source.

TABLE 1 S-II F-I F-III 2θ (°) I/I_(o) (%) 2θ (°) I/I_(o) (%) 2θ (°)I/I_(o) (%) 4.67 1.3 4.92 2.6 4.63 20.8 5.03 6 7.69 34.6 7.82 100 6.835.8 7.91 100 9.29 16.9 7.17 16.1 9.89 2.5 10.16 22.7 7.73 100 10.22 210.35 5.4 9.03 1.3 10.74 7.4 13.77 10.7 9.31 1.7 14.86 9.1 13.97 15.29.66 2.4 15.45 2.3 15.06 6.9 10.27 1.6 15.92 15.9 15.71 22.3 10.47 2.216.67 1.7 15.87 7.4 10.91 6.3 16.98 3.1 16.35 34.5 13.63 2.1 18.28 17.816.77 12.3 14.09 4.6 18.56 7 17.28 10 15.10 4.1 20.58 13.1 17.62 47.915.52 10.5 20.85 8.8 18.09 43.9 16.45 9.1 21.64 3.9 20.43 42 16.67 7.622.19 4.8 20.80 33.6 17.21 4.9 22.65 2.9 21.31 42.7 17.53 2.4 23.28 3.421.71 13 18.33 28.2 23.97 11.8 21.85 14.5 18.69 11.1 24.31 6.3 22.1312.8 19.37 3.5 25.52 3.9 22.26 16.3 20.29 8.6 26.20 3.4 23.51 13.2 20.6417.2 26.47 3.1 23.69 15.9 21.02 12.7 28.84 6.4 23.91 25.6 21.68 5.130.13 3.5 24.31 38.7 22.01 8.3 31.12 2.9 25.22 8 22.29 8 25.67 8.9 23.177.8 27.05 18.9 23.39 9.1 27.89 13.3 24.30 13.6 28.24 8.6 25.76 3.4 28.7121.3 26.05 4 29.89 8.9 26.63 5.5 30.24 18.7 27.01 3.1 30.88 5.8 27.492.8 31.44 7.6 28.10 1.8 33.06 4.5 28.73 10.9 34.36 6 29.42 3.2 30.00 3.730.89 2.1 31.34 2.4 31.70 1.1 32.81 1 32.91 0.8 33.48 2

Further Characterization of F-I and F-III

Hygroscopicicity studies were performed on F-I and F-III. The moisturesorption isotherms for F-I and F-III are shown in FIG. 4. Upon initialexposure of the samples to approximately 5% RH, there was an immediateweight gain of 1.5% and 1.7% moisture for F-I and F-III, respectively,equivalent to approximately 0.5 mole of water. Both forms show acontinuous sorption of moisture through the entire humidity range, whichis likely due to incorporation of water molecules in the lattices.

The difference in the moisture uptake of the two forms likely reflectsthe amount of water that can be incorporated into the two lattices(i.e., the amount of available space in the lattice that can accommodatewater molecules). Lack of hysteresis in the sorption-desorptionisotherms of F-I and F-III indicates that the crystal forms rapidlyequilibrate at any given humidity.

The moisture sorption profiles for F-I and F-III reveal that these formsare essentially non-stoichiometric hydrates. At ambient relativehumidity (about 50% RH), F-I contains approximately 1.7% water,corresponding to 0.5 moles of water, while F-III has sorbed about 3.0%water which corresponds to about 0.85 moles of water. The bulk forms ofF-I and F-III rapidly equilibrate with the atmosphere, so that the watercontent observed by analytical techniques is a reflection of therelative humidity at the time of data collection. Lot-to-lot differencesobserved in the DSC data likely results from the samples being hydratedto different extents due to different ambient storage conditions.

XRD patterns were obtained for samples of F-I and F-III stored atdifferent relative humidities (0, 22, 50, and 80%). There is a gradualshifting of the initial (0% RH) F-III peaks at about 13.8, 17.6, 18.0,20.5 and 24.0° in 2θ as well as slight shifting of less intense peaks,as the relative humidity is increased. These observed changes in the XRDpatterns of F-III indicate that the unit cell dimensions are changing,presumably to accommodate weakly held water molecules as the relativehumidity is increased. The continuous shifting of peaks with humiditycorrelates well with moisture sorption data that showed a gradual weightgain over this RH range, providing evidence for variable hydrateformation.

A similar experiment was carried out on F-I to determine whether varyingthe relative humidity would have a similar effect on its lattice (0, 25,52, 73 and 95% RH). Very slight shifting of the 0% RH peaks at about7.7, 18.3, 18.5, 20.5, 20.8° in 2θ is observed as the relative humidityis increased. The peaks at about 7.7, 20.8, and 24.1 also appear tobecome slightly broadened and less resolved at higher relativehumidities, indicating that water is being sorbed into amorphouscomponents (or plasticizes the solid), particularly at 73 and 95% RH.The shifting of peaks in the XRD patterns of F-I is less dramatic thanthe peak shifts observed as F-III was exposed to different relativehumidities. This suggests that the F-I lattice does not undergo the sameexpansion and/or contraction as the F-III lattice.

F-I and F-III were found to be stable over the entire relative humidityrange, despite the ability of F-III to sorb nearly twice as much water.The two forms were found to have comparable crystal size, morphology,aqueous solubilities and dissolution rates.

A drying study was carried out to monitor the desolvation of S-II as afunction of drying time and temperature (see FIG. 5). XRD patterns weretaken at various timepoints during the desolvation experiment. Manydiffraction peaks from the desolvation study of S-II appear at similarangles to F-I, confirming that the lattices of S-II and F-I are verysimilar. The disappearance of diffraction peaks at about 6.8, 7.2 and14.0° in 2θ after only minimal drying suggests that these reflectionsmay be attributed to crystallographic planes containing partial electrondensity of ethyl acetate molecules.

Extended annealing of the solvated material under vacuum at hightemperatures yielded F-I. F-I prepared this way showed a high degree ofcrystallinity by XRD. Therefore, material generated by crystallizationfrom a solution of ethanol and ethyl acetate followed by vacuum dryingfor only a few hours as taught in '474 showed very poor crystallinitybecause such a procedure results in partially desolvated S-II.

F-I and F-III have several advantages over the prior art form ofarzoxifene described above. Relative to the arzoxifene produced by theprocedures taught in '474, F-I and F-III are more stable at ambienttemperature and are, therefore, more amenable to pharmaceuticaldevelopment, i.e., development of a dosage formulation. In addition, F-Iand F-III are much more crystalline than the form disclosed in '474.Crystalline materials are generally less hygroscopic and more stable(e.g., less prone to chemical degradation, maintains consistent potency)than amorphous materials and are, therefore, more desirable forformulation processing. Furthermore, unlike the form of arzoxifeneproduced by the procedures taught in '474, which contained ethyl acetateand water in its lattice, F-I and F-III contain only water.

Characterization Methods

DSC measurements were performed on a TA Instruments 2920 Modulated DSCattached to a Thermal Analyst 3100 and equipped with a refrigeratedcooling system. Samples (3-5 mg) were heated in crimped aluminum pansfrom 10 to 240° C. at a heating rate of 2° C./min.

TGA analyses were performed on a TA Instruments 2050 ThermogravimetricAnalyzer attached to a Thermal Analyst 3100. Samples (5-10 mg) wereheated in open pans from 25° C. to 250° C. at a heating rate of 5°C./min.

XRD patterns were obtained on a Siemens D5000 X-ray powderdiffractometer, equipped with a CuKα source (λ=1.54056 Å) and a Kevexsolid-state detector, operating at 50 kV and 40 mA. Each sample wasscanned between 4° and 35° in 2θ. Samples were allowed to equilibratefor at least 30 minutes at the desired temperature and/or relativehumidity before data collection.

Hygroscopicity measurements were made for F-I and F-III using the VTImethod as follows. Each sample was dried under vacuum at 60° C. until nofurther weight loss was detected, at which time the sample chamber wasbrought to 0% relative humidity. Moisture sorption isotherms wereobtained at 25° C. using a VTI vacuum moisture balance with thefollowing conditions: sample size 10-15 mg, adsorption/desorption range0-95% relative humidity, step interval 5%, sample interval 10 minutes.

The following examples further illustrate processes for preparing thehydrate of the present invention. The examples are not intended to belimiting to the scope of these processes in any respect, and should notbe so construed.

EXAMPLE Example 1 F-III From6-Isopropoxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiopheneHydrochloride

To a methylene chloride solution (100 mL) of6-isopropoxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophenehydrochloride (10 g, 18 mmol) under a nitrogen atmosphere at −10° C. to−20° C., was added BCl_(3(g)) (4.23 g, 34 mmol) at a rate whichmaintains the temperature of the reaction below −10° C. After theaddition was complete, the reaction was allowed to stir for anadditional 2 hours. To the reaction, isopropyl alcohol (IPA, 12.35 mL,167 mmol) was slowly added at less than −10° C. and stirring wascontinued for 30 minutes. A separate flask was charged with 100 mL waterand cooled with an ice bath to approximately 0° C. The product solutionwas transferred to the water via cannula, maintaining vigorous stirring.The resultant white slurry was allowed to stir at 0° C. for 1 hour. Theproduct was recovered by filtration and rinsed with 25 mL 40%CH₂Cl₂/water then with 25 mL cold water. The product was suspended into60 mL IPA and 60 mL water and heated to 60° C. A solution was obtainedat 48° C. Additional water (120 mL) was added. The solution was allowedto cool to 35° C. and the slurry was further cooled slowly to 0-5° C.and stirred for several hours. The product was isolated by filtrationand washed with cold deionized water (25 mL). F-III wetcake was dried toa constant weight in vacuo at 50° C. for 12 to 24 hours to provideF-III.

Example 2 F-III From6-Benzyloxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene-(S-oxide)

To a 250 mL Parr bottle was added deionized water (5.25 mL), 1M HCl(7.74 mL, 7.75 mmol), 10% Pd/C (type A32110, 1.37 g, 1.29 mmol Pd),[6-benzyloxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene-(S-oxide)(3 g, 5.16 mmol), and isopropyl alcohol (32 mL) at ambient temperature.The bottle was fitted to a Parr shaker, sequentially evacuated andgassed with nitrogen twice, and subsequently evacuated and filled withhydrogen gas to a pressure of 30 psig. The shaker was started and thereaction mixture was heated to 60° C. The reaction was determined to becomplete by HPLC analysis after approximately 4 hours. The reactionmixture was filtered through a pad of diatomaceous earth, and the padwas washed with 0.1 M HCl (2×10 mL). The solvent was removed in vacuo atapproximately 50° C. The resultant residue was dissolved into 50%isopropyl alcohol/deionized water (30 mL) and gently heated on a steambath until a solution was obtained. To the solution was added deionizedwater (22 mL) and the solution was allowed to cool to ambienttemperature. The product slurry is further cooled to 0° C. The productwas isolated by filtration, washed with cold deionized water (2×15 mL),and dried in vacuo at 50° C. to constant weight to provide F-III.

Example 3 F-III from[6-Isopropoxy-3-[4-[2-(piperidin-1-yl)ethoxy)phenoxy]-2-(4-methoxyphenyl]benzo[b]thiopene-(S-oxide)

Methylene chloride (105 L) and[6-isopropoxy-3-[4-[2-(piperidin-1-yl)ethoxy)phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiopene-(S-oxide)(10.5 kg) are combined and cooled to −15 to −20° C. Boron trichloride(4.6 kg) is added while maintaining the reaction temperature between −10and −20° C. Stirring is continued until the area % by HPLC of thestarting material is less than 1%. HPLC system (4.6 mm ID×25 cm ZorbaxSB-Phenyl column 30° C., 70:30 methanol: 0.01 N sulfuric acid; flow 1.5ml/min; detector 300 nm). Isopropanol (10.28 kg) is added whilemaintaining the reaction temperature between −10 and −20° C. Thereaction mixture is stirred for 30 to 45 minutes. The crude reactionproduct is isolated by adding the reaction mixture to an additional 105L of water, pre-cooled to 2 to 7° C. while maintaining the reactiontemperature at 2 to 7° C. The addition is followed by a methylenechloride rinse (20 L) pre-cooled to 7 to 2° C. The crystalline slurry isstirred for 2 to 3 hours and filtered and sequentially washed withmethylene chloride (26 kg) pre-cooled to 7 to 2° C. and water (53 L)pre-cooled to 7 to 2° C. The crude wet cake is combined with water (42L) and isopropanol (40 L) and heated to 65 to 70° C. to effectdissolution. The hot solution is filtered. The filtration is followedwith a rinse consisting of a mixture of isopropanol (20 L) and water (21L) heated to 65 to 70° C. and a rinse consisting of water (126 L)pre-heated to 65 to 70° C. The solution is cooled to 40 to 45° C.,seeded and stirred at this temperature for 2 to 3 hours to allow crystalgrowth. The slurry is cooled to 0 to 5° C., stirred for 3 to 4 hours andfiltered. The filter cake is washed with water (122.6L) pre-cooled to 2to 7° C. The product is vacuum dried at a maximum temperature of 50° C.until the change in cake weight was less than 0.05 kgs over a 2-4 hourperiod. Yield: 8.468 kgs (87.3%). HPLC potency: 98.5%. Water by KarlFischer: 3.0%. Total related substances by HPLC: 1.79%.

Example 4 F-III from[6-Benzyloxy-3-[4-[2-(piperidin-1-yl)ethoxy)phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiopene-(S-oxide)

Tetrahydrofuran (261 ml), water (45 ml) concentrated sulfuric acid (6.14g) and [6-benzyloxy-3-[4-[2-(piperidin-1-yl)ethoxy)phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiopene-(S-oxide) (HPLCpotency 99%, HPLC total related substance level 0.35%) were combined andstirred until homogeneous. 10% Pd/C (5.6 g slurried in 22 ml of water)was added with a 5 ml water rinse. The resulting slurry was evacuatedand overlaid with 60 psi of hydrogen. The reaction temperature wasadjusted to 30° C. After 2 hours, 10% Pd/C (5.6 g) of was added withwater (30 ml). Hydrogenation at 60 psi and 30° C. was continued for anadditional 22 hours. An additional 4.40 g of 10% Pd/C in 30 ml water wasadded and hydrogenation at 60 psi and 30° C. continued for an additional2.5 hours. The catalyst was removed by filtration and the pH of thefiltrate was adjusted to 7.24 with 50% sodium hydroxide. Sodium chloride(8.66 g) dissolved in water (18 ml) was added and the biphasic solutionstirred for 30 minutes. The phases were separated and the aqueous phasewas back extracted with 50 ml of tetrahydrofuran. The organic phaseswere combined and concentrated by atmospheric distillation to a volumeof 50 ml. To the concentrate at 24° C. was added methanol, 180 ml over a1 hour period. The resulting crystalline slurry was stirred for 30minutes at 24° C., cooled to 0° C. and stirred for 1 hour. The solidswere isolated by filtration and washed sequentially with 39 ml of waterand 39 ml of methanol followed by vacuum drying overnight at 50° C.Yield 15.52 g (67.8%)

Isopropanol (33 ml), water (66 ml) and 10 g of solid isolated from abovewere combined. To the stirred mixture at 25° C. is added 1.8 M HCl (21ml). The solids quickly dissolved followed by re-precipitation of thehydrochloride salt. After stirring for 30 minutes, the slurry was heatedto 70° C. to effect dissolution of all of the solids. The solution wascooled to 60° C. and 33 ml of water added. The resulting solution wascooled to 25° C. over a 3 hour period during which time thehydrochloride salt precipitated. The slurry was stirred forapproximately 3 hours at 25° C., filtered, washed with water (30 ml) andvacuum dried overnight at 50° C. to yield 8.9 g (82.7%) of F-III. HPLCpotency: 96.5%. Water by Karl Fischer: 2.44%. HPLC related substances:1.09%.

Utilities

As used herein, the term “effective amount” means an amount of F-IIIthat is capable of inhibiting conditions, or detrimental effectsthereof, described herein. When F-III is co-administered with estrogen,progestin, an aromatase inhibitor, an LHRH analogue, or an AChEinhibitor, the term “effective amount” also means an amount of such anagent capable of producing its intended effect.

The terms “inhibiting” and “inhibit” include their generally acceptedmeaning, i.e., preventing, prohibiting, restraining, alleviating,ameliorating, slowing, stopping, or reversing the progression orseverity of a pathological condition, or sequela thereof, describedherein.

The terms “preventing”, “prevention of”, “prophylaxis”, “prophylactic”and “prevent” are used herein interchangeably and refer to reducing thelikelihood that the recipient of F-III will incur or develop any of thepathological conditions, or sequela thereof, described herein.

The terms “estrogen deprived” and “estrogen deprivation” refer to acondition, either naturally occurring or clinically induced, where awoman can not produce sufficient endogenous estrogenic hormones tomaintain estrogen dependent functions, e.g., menses, homeostasis of bonemass, neuronal function, cardiovascular condition, etc. Such estrogendeprived situations arise from, but are not limited to, menopause andsurgical or chemical ovarectomy, including its functional equivalent,e.g., medication with an aromatase inhibitor, GnRH agonists orantagonists, ICI 182780, and the like. Disease states associated with anestrogen deprived state include, but are not limited to: bone loss,osteoporosis, cardiovascular disease and hyperlipidemia.

As used herein, the term “estrogen” includes steroidal compounds havingestrogenic activity such as, for example, 17β-estradiol, estrone,conjugated estrogen (Premarin®), equine estrogen 17β-ethynyl estradiol,and the like. A preferred estrogen-based compound is Premarin®, andnorethylnodrel.

As used herein, the term “progestin” includes compounds havingprogestational activity such as, for example, progesterone,norethylnodrel, nongestrel, megestrol acetate, norethindrone, and thelike. Norethindrone is a preferred progestin-based agent.

As used herein the term “aromatase inhibitor” includes compounds capableof inhibiting aromatase, for example commercially available inhibitorssuch as aminoglutemide (CYTANDREN®), Anastrazole (ARIMIDEX®), Letrozole(FEMARA®), Formestane (LENATRON®), Exemestane (AROMASIN®), and the like.

As used herein, the term “LHRH analogue” refers to an analogue oflutenizing hormone releasing hormone that inhibits estrogen productionin a premenopausal women including for example, goserlin (ZOLADEX®),leuprolide (LUPRON®) and the like.

As used herein, the term “AChE inhibitor” includes compounds thatinhibit acetyl choline esterase, for example, physostigmine salicylate,tacrine hydrochloride, donepezil hydrochloride and the like.

The term “up-regulate ChAT” refers to increasing the enzymatic activityof ChAT, i.e., promoting the conversion of choline to acetyl choline.This promotion would include an increase in the efficiency and/or rateof reaction of ChAT and choline and/or an increase in the amount of ChATpresent at the site of action. This increase in the amount of enzymepresent may be due to gene regulation or other synthetic step of theenzyme's formation and/or a decrease in the enzyme's de-activation andmetabolism.

Selected Testing Procedures

General Rat Preparation Procedure: Seventy-five day old (unlessotherwise indicated) female Sprague Dawley rats (weight range of 200 to225 g) are obtained from Charles River Laboratories (Portage, Mich.).The animals are either bilaterally ovariectomized (OVX) or exposed to aSham surgical procedure at Charles River Laboratories, and then shippedafter one week. Upon arrival, they are housed in metal hanging cages ingroups of 3 or 4 per cage and have ad libitum access to food (calciumcontent approximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. Thephotoperiod in the room was 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection: After a one week acclimation period(therefore, two weeks post-OVX) daily dosing with F-III is initiated.17α-ethynyl estradiol or F-III is given orally, unless otherwise stated,as a suspension in 1% carboxymethylcellulose or dissolved in 20%cyclodextrin. Animals are dosed daily for 4 days. Following the dosingregimen, animals are weighed and anesthetized with a ketamine:Xylazine(2:1, v:v) mixture and a blood sample is collected by cardiac puncture.The animals are then sacrificed by asphyxiation with CO₂, the uterus isremoved through a midline incision, and a wet uterine weight isdetermined. 17α-ethynyl estradiol is obtained from Sigma Chemical Co.,St. Louis, Mo.

Cardiovascular Disease/Hyperlipidemia

The blood samples from above are allowed to clot at room temperature for2 hours, and serum is obtained following centrifugation for 10 minutesat 3000 rpm. Serum cholesterol is determined using a Boehringer MannheimDiagnostics high performance cholesterol assay. Briefly the cholesterolis oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogenperoxide is then reacted with phenol and 4-aminophenazone in thepresence of peroxidase to produce a p-quinone imine dye, which is readspectrophotemetrically at 500 nm. Cholesterol concentration is thencalculated against a standard curve. The entire assay is automated usinga Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay

The uteri from above are kept at 4° C. until time of enzymatic analysis.The uteri are then homogenized in 50 volumes of 50 mM Tris buffer(pH−8.0) containing 0.005% Triton X-100. Upon addition of 0.01% hydrogenperoxide and 10 mM O-phenylenediamine (final concentrations) in Trisbuffer, increase in absorbance is monitored for one minute at 450 nm.The presence of eosonophils in the uterus is an indication of estrogenicactivity of a compound. The maximal velocity of a 15 second interval isdetermined over the initial, linear portion of the reaction curve.

Inhibition of Bone Loss (Osteoporosis) Test Procedure

Following the general preparation procedure described above, the ratsare treated daily for thirty-five days (6 rats per treatment group) andsacrificed by carbon dioxide asphyxiation on the 36th day. Thethirty-five day time period is sufficient to allow maximal reduction inbone density, measured as described herein. At the time of sacrifice,the uteri are removed, dissected free of extraneous tissue, and thefluid contents are expelled before determination of wet weight in orderto confirm estrogen deficiency associated with complete ovariectomy.Uterine weight is routinely reduced about 75% in response toovariectomy. The uteri are then placed in 10% neutral buffered formalinto allow for subsequent histological analysis.

The right femurs are excised and digitilized X-rays generated andanalyzed by an image analysis program (NIH image) at the distalmetaphysis. The proximal aspect of the tibiae from these animals arealso scanned by quantitative computed tomography. In accordance with theabove procedures, F-III or ethynyl estradiol (EE₂) in 20% hydroxypropylβ-cyclodextrin are orally administered to test animals. F-III is alsouseful in combination with estrogen or progestin.

MCF-7 Proliferation Assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.)supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM), HEPES{(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]10 mM},non-essential amino acids and bovine insulin (1 ug/mL) (maintenancemedium). Ten days prior to assay, MCF-7 cells are switched tomaintenance medium supplemented with 10% dextran coated charcoalstripped fetal bovine serum (DCC-FBS) assay medium) in place of 10% FBSto deplete internal stores of steroids. MCF-7 cells are removed frommaintenance flasks using cell dissociation medium (Ca++/Mg++ free HBSS(phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA). Cellsare washed twice with assay medium and adjusted to 80,000 cells/mL.Approximately 100 mL (8,000 cells) are added to flat-bottom microculturewells (Costar 3596) and incubated at 37° C. in a 5% CO₂ humidifiedincubator for 48 hours to allow for cell adherence and equilibrationafter transfer. Serial dilutions of drugs or DMSO as a diluent controlare prepared in assay medium and 50 mL transferred to triplicatemicrocultures followed by 50 mL assay medium for a final volume of 200mL. After an additional 48 hours at 37° C. in a 5% CO₂ humidifiedincubator, microcultures are pulsed with tritiated thymidine (1uCi/well) for 4 hours. Cultures are terminated by freezing at −70° C.for 24 hours followed by thawing and harvesting of microcultures using aSkatron Semiautomatic Cell Harvester. Samples are counted by liquidscintillation using a Wallac BetaPlace β counter.

DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Ind. Atabout 55 days of age, the rats receive a single oral feeding of 20 mg of7,12-dimethylbenz[a]anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpated at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

F-III is administered either through intraperitoneal injections in 2%acacia, or orally. Orally administered compounds are either dissolved orsuspended in 0.2 mL corn oil. Each treatment, including acacia and cornoil control treatments, is administered once daily to each test animal.Following the initial tumor measurement and selection of test animals,tumors are measured each week by the above-mentioned method. Thetreatment and measurements of animals continue for 3 to 5 weeks at whichtime the final areas of the tumors are determined. For each compound andcontrol treatment, the change in the mean tumor area is determined.

Uterine Fibrosis Test Procedures

Test 1: Between 3 and 20 women having uterine fibrosis are administeredF-III. The amount of compound administered is from 0.1 to 1000 mg/day,and the period of administration is 3 months. The women are observedduring the period of administration, and up to 3 months afterdiscontinuance of administration, for effects on uterine fibrosis.

Test 2: The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3: The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4: Prolonged estrogen stimulation is used to induce leiomyomata insexually mature female guinea pigs. Animals are dosed with estradiol 3-5times per week by injection for 2-4 months or until tumors arise.Treatment consisting of F-III or vehicle is administered daily for 3-16weeks and then animals are sacrificed and the uteri harvested andanalyzed for tumor regression.

Test 5: Tissue from human leiomyomas are implanted into the peritonealcavity and/or uterine myometrium of sexually mature, castrated, female,nude mice. Exogenous estrogen is supplied to induce growth of theexplanted tissue. In some cases, the harvested tumor cells are culturedin vitro prior to implantation. Treatment consisting of F-III or vehicleis supplied by gastric lavage on a daily basis for 3-16 weeks andimplants are removed and measured for growth or regression. At the timeof sacrifice, the uteri are harvested to assess the status of the organ.

Test 6: Tissue from human uterine fibroid tumors is harvested andmaintained, in vitro, as primary non-transformed cultures. Surgicalspecimens are pushed through a sterile mesh or sieve, or alternatelyteased apart from surrounding tissue to produce a single cellsuspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, F-III, and vehicle. Levels of steroidhormone receptors are assessed weekly to determine whether importantcell characteristics are maintained in vitro. Tissue from 5-25 patientsis utilized.

Test 7: F-III's ability to inhibit estrogen-stimulated proliferation ofleiomyoma-derived ELT cell lines is measured substantially as describedin Fuchs-Young, et al., “Inhibition of Estrogen-Stimulated Growth ofUterine Leiomyomas by Selective Estrogen Receptor Modulators”, Mol.Car., 17(3): 151-159 (1996), the teachings of which are hereinincorporated.by reference.

Endometriosis Test Procedures

Test 1: Twelve to thirty adult CD strain female rats are used as testanimals. They are divided into three groups of equal numbers. Theestrous cycle of all animals is monitored. On the day of proestrus,surgery is performed on each female. Females in each group have the leftuterine horn removed, sectioned into small squares, and the squares areloosely sutured at various sites adjacent to the mesenteric blood flow.In addition, females in Group 2 have the ovaries removed. On the dayfollowing surgery, animals in Groups 1 and 2 receive intraperitonealinjections of water for 14 days whereas animals in Group 3 receiveintraperitoneal injections of 1.0 mg of F-III per kilogram of bodyweight for the same duration. Following 14 days of treatment, eachfemale is sacrificed and the endometrial explants, adrenals, remaininguterus, and ovaries, where applicable, are removed and prepared forhistological examination. The ovaries and adrenals are weighed.

Test 2: Twelve to thirty adult CD strain female rats are used as testanimals. They are divided into two equal groups. The estrous cycle ofall animals is monitored. On the day of proestrus, surgery is performedon each female. Females in each group have the left uterine hornremoved, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow.Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of F-III perkilogram of body weight for the same duration. Following 21 days oftreatment, each female is sacrificed and the endometrial explants andadrenals are removed and weighed. The explants are measured as anindication of growth. Estrous cycles are monitored.

Test 3: Autographs of endometrial tissue are used to induceendometriosis in rats and/or rabbits. Female animals at reproductivematurity undergo bilateral oophorectomy, and estrogen is suppliedexogenously thus providing a specific and constant level of hormone.Autologous endometrial tissue is implanted in the peritoneum of 5-150animals and estrogen supplied to induce growth of the explanted tissue.Treatment consisting of a compound of the present invention is suppliedby gastric lavage on a daily basis for 3-16 weeks, and implants areremoved and measured for growth or regression. At the time of sacrifice,the intact horn of the uterus is harvested to assess status ofendometrium.

Test 4: Tissue from human endometrial lesions is implanted into theperitoneum of sexually mature, castrated, female, nude mice. Exogenousestrogen is supplied to induce growth of the explanted tissue. In somecases, the harvested endometrial cells are cultured in vitro prior toimplantation. Treatment consisting of F-III supplied by gastric lavageon a daily basis for 3-16 weeks, and implants are removed and measuredfor growth or regression. At the time of sacrifice, the uteri areharvested to assess the status of the intact endometrium.

Test 5: Tissue from human endometrial lesions is harvested andmaintained in vitro as primary non-transformed cultures. Surgicalspecimens are pushed through a sterile mesh or sieve, or alternatelyteased apart from surrounding tissue to produce a single cellsuspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, F-III, and vehicle. Levels of steroidhormone receptors are assessed weekly to determine whether importantcell characteristics are maintained in vitro. Tissue from 5-25 patientsis utilized.

CNS Disorders Including Alzheimer's Disease

Estrogens, such. as 17β-estradiol, regulate gene transcription bybinding to estrogen receptors (ER) which reside in the cytoplasm ofcertain cell populations. Ligand activation of the ER is a prerequisitefor nuclear transport of the complex where binding to a 13 base-pairpalindromic DNA consensus sequence (estrogen response element, or ERE)begins assembly of a transcriptional apparatus which culminates in theactivation of appropriate target genes. A variety of genes have beenidentified which are regulated by estrogen. These include cytoskeletalproteins, neuro-transmitter biosynthetic and metabolic enzymes andreceptors, as well as other hormones and neuropeptides. ERE's have beenidentified in many estrogen-responsive genes including vitellogenin,c-fos, prolactin, and luteinizing hormone.

Of significance in the central nervous system, ERE-like sequences havebeen identified in p75^(ngr) and trkA, both of which serve as signalingmolecules for the neurotrophins: nerve growth factor (NGF), brainderived nerve growth factor (BDNGF), and neurotrophin-3.

BDNF as well as NGF have been shown to promote the survival ofcholinergic neurons in culture. It is postulated that if theinteractions between neurotrophins and estrogens are important for thedevelopment and survival of basal forebrain neurons (which degenerate inAlzheimer's disease) then clinical conditions in which an estrogendeficiency exists (as after menopause) may contribute to a loss of theseneurons.

The following experiment is conducted in ovariectomized rats (preparedas described above) to determine the similarities and/or differencesbetween F-III and estrogen at affecting gene expression in various brainregions. Six week old rats are dosed daily with subcutaneous injectionsof estradiol benzoate (0.03 mg/kg), F-III or vehicle (control). Afterfive weeks of treatment, animals are sacrificed and their brains removedand hippocampi collected by microdissection. The hippocampi are fastfrozen in liquid nitrogen and stored at −70° C. Total RNA is preparedfrom pooled tissue from the appropriate treatment and control groups andreverse transcribed using a 3′ oligonucleotide primer which is selectedfor specific MRNA (poly−A+) populations. Polymerase chain reactions(PCR) are carried out in a cocktail consisting of: random 5′oligonucleotides (10 base-pairs in length; total of 150), reactionbuffer, Taq polymerase, and a ³²PdTCP.

After 40 rounds of amplification, the reaction products are sizefractionated on a 6% TBE-urea gel, dried and exposed to X-ray film. Theresulting mRNA display patterns are compared between treatment groups.

Use of F-III in Conjunction with Estrogen

Peri- and post-menopausal women often undergo hormone replacementtherapy (HRT) to combat negative consequences associated with the dropin circulating endogenous estrogen, e.g., to treat hot flashes. However,HRT has been associated with increased risks of certain cancersincluding uterine and breast cancer. F-III may be employed inconjunction with HRT to inhibit these risks.

Use of F-III in Conjunction With an Aromatase Inhibitor

By definition, the ovaries of a postmenopausal woman are notfunctioning. Her only source of estrogen is through conversion ofadrenal androgens to estrogens by the enzyme aromatase, which is foundin peripheral tissues (including fat, muscle and the breast tumoritself). Thus, drugs that inhibit aromatase (aromatase inhibitors)deplete the postmenopausal woman of circulating estrogen. Estrogendeprivation by means of aromatase inhibition is an important treatmentoption for patients with metastatic breast cancer. During therapy withan aromatase inhibitor, lack of circulating estrogen may cause negative,unintended side-effects, for example on serum lipid levels. F-III may beemployed to inhibit these negative effects.

Use of F-III in Conjunction with a LHRH Analogue

Continuous exposure to a LHRH (lutenizing hormone releasing hormone)analogue inhibits estrogen production in the premenopausal women bydesensitizing the pituitary gland, which then no longer stimulates theovaries to produce estrogen. The clinical effect is a “medicaloophrectomy” which is reversible upon cessation of the LHRH analogue.During therapy with a LHRH analogue, lack of circulating estrogen maycause negative, unintended side-effects, for example on serum lipidlevels. F-III may be employed to inhibit these negative effects.

Increasing Levels of Acetyl Choline

It is known that patients suffering from Alzheimer's disease have amarkedly smaller level of cholinergic neurons in the hippocampus thantheir non-Alzheimer peers. The progressive loss of these cholinergicneurons appears to mirror the progressive loss in memory and cognitivefunction in these patients. It is thought that one reason for thedecline of these neurons is the loss or decreased function of theneurotransmitter, acetyl choline.

The level of acetylcholine in a neuron is basically determined by wherethe equilibrium between its bio-synthesis and bio-degradation lies. Theenzyme choline acetyltransferase (ChAT) is primarily responsible for itssynthesis and acetylcholineesterase (AChE) for its degradation.

In the order to determine F-III's effect on levels of ChAT, thefollowing experiment is performed: Following the general rat preparationprocedure described above, 40 rats are dosed daily by subcutaneousinjection or oral gavage with F-III at 3 mg/kg/day in a vehiclecontaining 10% cyclodextrin, estradiol benzoate at 0.03 or 0.3mg/kg/day, or vehicle control. Animals are treated for 3 or 10 days.There are twenty animals per each dosing regimen. At the appropriatetime intervals, the animals are sacrificed and their brains dissected.The particular portions of the brains are homogenized and assayed.Homogenates from the hippocampus and frontal cortex were processed anddetermination of ChAT activity is made by a radio-labelled assay of thebio-synthesis of acetyl choline. This procedure may be found in Schoeppet al., J. Neural Transmiss., 78:183-193, 1989, the teachings of whichare incorporated by reference.

As expected, in the OVX animals, ChAT levels are reduced >50% (p<0.001)compared to the sham operated controls.

In another embodiment of the present invention, F-III is used incombination with an AChE inhibitor. Use of an AChE inhibitor increaseslevels of acetylcholine by blocking its degradation via inhibition ofAChE.

Benign Prostatic Hyperplasia (BPH)

For background on the link between estrogen action and treatment of BPHand prostate carcinoma, see PCT Application No. WO 98/07274,International Publication Date: Oct. 15, 1998.

In the experiments described below, the ability of F-III to bind atestrogen receptors in several human prostatic cancer cell lines isevaluated.

Lysates of the LNCaP, DU-45 and PC-3 human prostatic cancer cell linesare prepared in a TEG medium comprising 50 nM Tris·HCl pH 7.4, 1.5 mMethylenediamine tetraacetic acid (EDTA) 0.4 M KCl, 10% glycerol, 0.5 mM2-ME, and 10 mM sodium molybdate further containing the proteaseinhibitors pepstatin (1 mg/mL), leupeptin (2 mg/mL), aprotinin (5 mg/mL)and phenylmethylsulfonyl fluoride (PMSF, 0.1 mM) (TEGP).

The cell lysates are centrifuged and the pellets resuspended in coldTEGP (1 mL TEGP/100 mg of pellet) and sonicated for 30 seconds (dutycycle 70%, output 1.8) on a Branson Model 450 Sonifier. Lysates arepelleted by centrifugation at 10,000×G for 15 minutes at 4° C. afterwhich the supernates are withdrawn and either used immediately or storedat −70° C.

Competitive Binding Assay: The binding buffer is TEG in which the 0.4 MKCl is replaced by 50 mM NaCl and to which 1 mg/mL of ovalbumin had beenfurther added (TEGO). F-III is diluted to 20 nM in TEGO from which3-fold serial dilutions are prepared. Assays are performed inround-bottom polyprolylene microplates in triplicate microwells. Eachwell receives 35 mL of tritiated 17β-estradiol (0.5 nM, specificactivity 60.1 Ci/mmol, DuPont-New England Nuclear, Boston, Mass.) and 35mL of cold competitot test compound (0.1 nM-5 mM) or TEGO, and followingincubation for 5 minutes at 4° C. with shaking, 70 mL of MCF-7 cell linelysate.

Plates are incubated for 24 hours at 4° C. after which time 70 mL ofdextran-coated charcoal (DCC) is added to each well followed by vigorousshaking for 8 minutes at 4° C. The plates are then centrifuged at 1500×Gfor 10 minutes at 4° C. Supernate is harvested from each well into aflexible polystyrene microplate for scintillation counting in a WallacMicobeta Model 1450 counter. Radioactivity is expressed asdisintegrations per minute (DPM) after correcting for countingefficiency (35-40%) and background. Additional controls are total countsand total counts+DCC to defined the lower limit of DCC extractablecounts. The results of these competitive binding assays are expressed asmean percent bound (% Bound) +/− standard deviation using the formula:${\% \quad {Bound}} = {\frac{{DPM}_{{test}\quad {compound}} - {DPM}_{{{total}\quad {count}}\quad + {DCC}}}{{DPM}_{{no}\quad {test}\quad {compound}} - {DPM}_{{{total}\quad {count}}\quad + {DCC}}} \times 100}$

Prevention of Breast Cancer

This invention also relates to the administration of F-III to arecipient who is at risk of developing de novo breast cancer. The term“de novo”, as used herein, means the lack of transformation ormetamorphosis of normal breast cells to cancerous or malignant cells inthe first instance. Such a transformation may occur in stages in thesame or daughter cells via an evolutionary process or may occur in asingle, pivotal event. This de novo process is in contrast to themetastasis, colonization, or spreading of already transformed ormalignant cells from the primary tumor site to new locations.

A person who is at no particular risk of developing breast cancer is onewho may develop de novo breast cancer, has no evidence or suspicion ofthe potential of the disease above normal risk, and who has never had adiagnosis of having the disease. The greatest risk factor contributingto the development of breast carcinoma is a personal history ofsuffering from the disease, or an earlier occurrence of the disease,even if it is in remission with no evidence of its presence. Anotherrisk factor is family history of the disease.

Induction of mammary tumors in rats by administration of the carcinogenN-nitroso-N-methylurea is a well-accepted animal model for the study ofbreast cancer and has been found suitable for analyzing the effect ofchemopreventive agents.

In two separate studies, 55-day old female Sprague-Dawley rats are givenan intravenous (Study 1) or intraperitoneal (Study 2) dose of 50 mg ofN-nitroso-N-methylurea per kilogram of body weight one week prior tofeeding ad libitum a diet into which varying amounts of F-III,(Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine base(tamoxifen base), or control are blended.

In Study 1, the dietary doses of 60 mg/kg of diet and 20 mg/kg of diettranslates into roughly comparable doses of 3 and 1 mg/kg of body weightfor the test animals.

In Study 2, the dietary doses of 20, 6, 2, and 0.6 mg/kg of diettranslates roughly into comparable doses of 1, 0.3, 0.1 and 0.03 mg/kgof body weight for the test animals.

Rats are observed for evidence of toxicity and are weighed and palpatedfor tumor formation once a week. The animals are sacrificed afterthirteen weeks (Study 1) or eighteen weeks (Study 2) and tumors areconfirmed and weighed at autopsy.

Formulations

The term “pharmaceutical” when used herein as an adjective meanssubstantially non-deleterious to the recipient mammal. By“pharmaceutical formulation” it is meant the carrier, diluent,excipients and active ingredient(s) must be compatible with the otheringredients of the formulation, and not deleterious to the recipientthereof.

F-III is preferably formulated prior to administration. The selection ofthe formulation should be decided by the attending physician taking intoconsiderations the same factors involved with determining the effectiveamount.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. Preferably, no more than two activeingredients are contained in said formulation. That is, it is preferredto formulate F-III with a second active ingredient selected from anestrogen, progestin, aromatase inhibitor, LHRH analogue and AChEinhibitor. Most preferred formulations are those where F-III is the soleactive ingredient.

Pharmaceutical formulations of the present invention are prepared byprocedures known in the art using well known and readily availableingredients. For example, F-III, either alone, or in combination with anestrogen, progestin, aromatase inhibitor, LHRH analogue, or an AChEinhibitor compound, are formulated with common excipients, diluents, orcarriers, and formed into tablets, capsules, suspensions, solutions,injectables, aerosols, powders, and the like.

Pharmaceutical compositions of this invention for parenteraladministration comprise sterile aqueous or non-aqueous solutions,dispersions, suspensions, or emulsions, as well as sterile powders whichare reconstituted immediately prior to use into sterile solutions orsuspensions. Examples of suitable sterile aqueous and non-aqueouscarriers, diluents, solvents or vehicles include water, physiologicalsaline solution, ethanol, polyols (such as glycerol, propylene glycol,poly(ethylene glycol), and the like), and suitable mixtures thereof,vegetable oils (such as olive oil), and injectable organic esters suchas ethyl oleate. Proper fluidity is maintained, for example, by the useof coating materials such as lecithin, by the maintenance of properparticle size in the case of dispersions and suspensions, and by the useof surfactants.

Parenteral compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms is ensured by theinclusion of antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of injectable formulations may bebrought about by the inclusion of agents which delay absorption such asaluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug following subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline material of low water solubility or bydissolving or suspending the drug in an oil vehicle. In the case of thesubcutaneous or intramuscular injection of a suspension containing aform of the drug with low water solubility, the rate of absorption ofthe drug depends upon its rate of dissolution.

Injectable “depot” formulations of F-III are made by formingmicroencapsulated matrices of the drug in biodegradable polymers such aspoly(lactic acid), poly(glycolic acid), copolymers of lactic andglycolic acid, poly (orthoesters), and poly (anhydrides) these materialswhich are described in the art. Depending upon the ratio of drug topolymer and the characteristics of the particular polymer employed, therate of drug release can be controlled.

Injectable formulations are sterilized, for example, by filtrationthrough bacterial-retaining filters, or by presterilization of thecomponents of the mixture prior to their admixture, either at the timeof manufacture or just prior to administration (as in the example of adual chamber syringe package).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, F-III is mixedwith at least one inert, pharmaceutical carrier such as sodium citrate,or dicalcium phosphate, and/or (a) fillers or extenders such asstarches, sugars including lactose and glucose, mannitol, and silicicacid, (b) binding agents such as carboxymethyl-cellulose and othercellulose derivatives, alginates, gelatin, poly(vinylpyrrolidine),sucrose and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar-agar, calcium carbonate, sodium bicarbonate, potatoor tapioca starch, alginic acid, silicates and sodium carbonate, (e)moisturizing agents such as glycerol; (f) solution retarding agents suchas paraffin, (g) absorption accelerating agents such as quaternaryammonium compounds, (h) wetting agents such as cetyl alcohol andglycerin monostearate, (i) absorbents such as kaolin and bentonite clay,and (j) lubricants such as talc, calcium stearate, magnesium stearate,solid poly(ethylene glycols), sodium lauryl sulfate, and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso contain buffering agents.

Solid compositions of a similar type may also comprise the fill in softor hard gelatin capsules using excipients such as lactose as well ashigh molecular weight poly(ethylene glycols) and the like.

Solid dosage forms such as tablets, dragees, capsules, pills andgranules can also be prepared with coatings or shells such as entericcoatings or other coatings well known in the pharmaceutical formulatingart. The coatings may contain opacifying agents or agents which releasethe active ingredient(s) in a particular part of the digestive tract, asfor example, acid soluble coatings for release of the activeingredient(s) in the stomach, or base soluble coatings for release ofthe active ingredient(s) in the intestinal tract.

The active ingredient(s) may also be microencapsulated in asustained-release coating, with the microcapsules being made part of apill of capsule formulation.

Liquid dosage forms for oral administration of F-III include solution,emulsions, suspensions, syrups and elixirs. In addition to the activecomponents, liquid formulations may include inert diluents commonly usedin the art such as water or other pharmaceutical solvents, solubilizingagents and emulsifiers such as ethanol, isopropanol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide, oils (in particular,cottonseed, ground nut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, poly(ethylene glycols), fatty acidesters of sorbitol, and mixtures thereof.

Besides inert diluents, the liquid oral formulations may also includeadjuvants such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Liquid suspension, in addition to the active ingredient(s) may containsuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite clay, agar-agar, andtragacanth, and mixtures thereof.

Compositions for rectal or intravaginal administration are prepared bymixing F-III with suitable non-irritating excipients such as cocoabutter, polyethylene glycol or any suppository wax which is a solid atroom temperature, but liquid at body temperature and therefore melt inthe rectum or vaginal cavity to release the active component(s). Thecompounds are dissolved in the melted wax, formed into the desiredshape, and allowed to harden into the finished suppository formulation.

F-III may also be administered in the form of liposomes. As is know inthe art, liposomes are generally derived from phospholipids or otherlipid substances. Lipososome formulations are formed by mono- ormultilamellar hydrated liquid crystals which are dispersed in an aqueousmedium. Any non-toxic, pharmaceutical, and metabolizable lipid capableof forming liposomes can be used. The present compositions in liposomeform can contain, in addition to F-III, stabilizers, excipients,preservatives, and the like. The preferred lipids are phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.

Methods for forming liposomes are know in the art as described, forexample, in Prescott, Ed., Methods in Cell Biology, Volume XIV, AcademicPress, New York, N.Y. (1976), p. 33 et seq.

The following formulation examples are illustrative only and are notintended to limit the scope of the present invention.

Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared usingthe following: Ingredient Quantity (mg/capsule) F-III 0.1-1000 Starch,NF   0-650 Starch flowable powder   0-650 Silicone fluid 350 centistokes  0-15

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2: Tablets Ingredient Quantity (mg/tablet) F-III  2.5-1000 Cellulose, microcrystalline 200-650 Silicon dioxide, fumed  10-650Stearate acid  5-15

The components are blended and compressed to form tablets.

Formulation 3: Tablets containing approximately 10 and 50 mgs,respectively, of F-III may be prepared as follows: Quantity QuantityIngredient (mg/tablet) (mg/tablet) F-III 11.3 56.5 Lactose Anhydrous176.8 128.2 Lactose Spray Dried Special 44.2 32.0 Povidone 11.0 13.0Polysorbate 80 2.5 2.6 Crosspovidone (Inside) 6.25 6.24 Crosspovidone(Outside) 6.25 6.5 Magnesium Stearate 1.5 1.7 Microcrystalline Cellulose0.0 13.0 (Outside)

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of F-III are made upas follows:

Formulation 4: Tablets Ingredient Quantity (mg/tablet) F-III 25-1000Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as 10%solution in water) Sodium carboxymethyl cellulose 4.5 Magnesium stearate0.5 Talc 1

F-III, starch, and cellulose are passed through a No. 45 mesh U.S. sieveand mixed thoroughly. The solution of polyvinylpyrrolidone is mixed withthe resultant powders which are then passed through a No. 14 mesh U.S.sieve. The granules so produced are dried at 50°-60° C. and passedthrough a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch,magnesium stearate, and talc, previously passed through a No. 60 U.S.sieve, are then added to the granules which, after mixing, arecompressed on a tablet machine to yield tablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 5: Suspensions Ingredient Quantity (mg/5 ml) F-III 0.1-1000mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mg Benzoic acidsolution 0.10 mL Flavor q.v. Color q.v. Purified water to   5 mL

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 6: Aerosol Quantity (% by Ingredient weight) F-III 0.25Ethanol 25.75 Propellant 22 (Chlorodifluoromethane) 70.00

F-III is mixed with ethanol and the mixture added to a portion of thepropellant 22, cooled to 30° C., and transferred to a filling device.The required amount is then fed to a stainless steel container anddiluted with the remaining propellant. The valve units are then fittedto the container.

Suppositories are prepared as follows:

Formulation 7: Suppositories Ingredient Quantity (mg/suppository) F-III250 Saturated fatty acid 2,000 glycerides

F-III is passed through a No. 60 mesh U.S. sieve and suspended in thesaturated fatty acid glycerides previously melted using the minimalnecessary heat. The mixture is then poured into a suppository mold ofnominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 8: Intravenous Solution Ingredient Quantity F-III   25 mgIsotonic saline 1,000 mL

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

Formulation 9: Combination Capsule I Ingredient Quantity (mg/capsule)F-III 50 Premarin 1 Avicel pH 101 50 Starch 1500 117.50 Silicon Oil 2Tween 80 0.50 Cab-O-Sil 0.25

Formulation 10: Combination Capsule II Ingredient Quantity (mg/capsule)F-III 50 Norethylnodrel 5 Avicel pH 101 82.50 Starch 1500 90 Silicon Oil2 Tween 80 0.50

Formulation 11: Combination Tablet Ingredient Quantity (mg/capsule)F-III 50 Premarin 1 Corn Starch NF 50 Povidone, K29-32 6 Avicel pH 10141.50 Avicel pH 102 136.50 Crospovidone XL10 2.50 Magnesium Stearate0.50 Cab-O-Sil 0.50

Dosage

The specific dose of F-III administered according to this invention isdetermined by the particular circumstances surrounding each situation.These circumstances include, the route of administration, the priormedical history of the recipient, the pathological condition or symptombeing treated, the severity of the condition/symptom being treated, andthe age and sex of the recipient.

Generally, an effective minimum daily dose of F-III is about 1, 5, 10,15, or 20 mg. Typically, an effective maximum dose is about 800, 100,60, 50, or 40 mg. Most typically, the dose ranges between 15 mg and 60mg. The exact dose may be determined, in accordance with the standardpractice in the medical arts of “dose titrating” the recipient; that is,initially administering a low dose of the compound, and graduallyincreasing the does until the desired therapeutic effect is observed.

Although it may be necessary to dose titrate the recipient with respectto the combination therapies discussed above, typical doses of activeingredients other than F-III are as follows: ethynyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), conjugated estrogenic hormones(e.g., Premarin®, Wyeth-Ayerst; 0.3-2.5 mg/day), medroxyprogesterone(2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), nonethindrone(0.5-2.0 mg/day), aminoglutemide (250-1250 mg/day, preferably 250 mgfour times per day), anastrazole (1-5 mg/day, preferably 1 mg once perday), letrozole (2.5-10 mg/day, preferably 2.5 mg once a day),formestane (250-1250 mg per week, preferably 250 mg twice weekly),exemestane (25-100 mg/day, preferably 25 mg once per day), goserlin(3-15 mg/three months, preferably 3.6-7.2 mg once every three months)and leuprolide (3-15 mg/month, preferably 3.75-7.5 mg once every month).

Route of administration

F-III can be administered by a variety of routes including oral, rectal,transdermal, subcutaneus, intravenous, intramuscular, and intranasal.The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. F-III, alone or incombination with estrogen, progestin, or an AChE inhibitor generallywill be administered in a convenient formulation.

The pharmaceutical compositions of this invention may be administered tohumans and other mammals (e.g., dogs, cats, horses, swine and the like)orally, rectally, intravaginally, parenterally, topically, bucally orsublingually, or nasally. The term “parenteral administration” refersherein to modes of administration which include intravenous,intramuscular, intraperitoneal, instrasternal, subcutaneous, orintraarticular injection or infusion.

Mode/Length of Administration

For the majority of the methods of the present invention, F-III isadministered continuously, from 1 to 3 times daily or as often as neededto deliver an effective amount of F-III to the recipient. Cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease. In the caseof restenosis, therapy may be limited to short (1-6 months) intervalsfollowing medical procedures such as angioplasty.

We claim:
 1. Crystalline 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride hydrate (F-III) having an X-ray diffraction pattern which comprises the following peaks: 4.6±0.2, 7.8±0.2, 9.3±0.2, 14.0±0.2, 17.6±0.2, 20.8±0.2, and 24.3±0.2° in 2θ; when obtained at 25±2° C. and 35±10% relative humidity from a copper radiation source (CuKα; λ=1.54056 Å).
 2. A pharmaceutical formulation comprising the crystalline compound of claim 1 and one or more pharmaceutical carriers, diluents, or excipients.
 3. A pharmaceutical formulation comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents, or excipients; and estrogen.
 4. A process for preparing a compound of claim 1 which comprises crystallizing 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride from a mixture of isopropanol and water. 